Project Summary In many infections (e.g., HIV or influenza), the preimmune repertoire may include few or no broadly neutralizing antibodies against critical epitopes. We seek to understand why some epitope reactivities are rare, and others more common. Complementarity determining region 3 of the heavy (H) chain (CDR-H3) lies at the center of the antigen binding site. It is the direct product of VDJ rearrangement and N addition. It is thus the focus for preimmune repertoire diversification. The first Ig quality control checkpoint tests whether nascent ? H chain (?HC) will associate with VpreB and ?5 to create a functional preBCR. Our premise is that preBCR selection of the preimmune repertoire restricts subsequent BCR epitope recognition and B cell antibody production. We examined the CDR-H3 repertoires of early and late preB cells from DH-altered mice, and then of living and apoptotic early and late preB cells of WT mice. PreBCR checkpoint passage was associated with selection for tyrosine (Y) at CDR-H3 101. The hydroxyl group of Y101 is positioned for multiple interactions with VpreB CDR-H3 sensing site amino acids R51, D57, and R101 (RDR). We will test the hypothesis that the CDR-H3 sensing site of VpreB creates an invariant antigenic epitope that selects particular CDR-H3 amino acids and thus shapes the binding characteristics of the antibody repertoire. For this developmental R21 application, we propose to test the role of VpreB RDR in controlling the prevalence of particular amino acids in CDR-H3 and to establish model systems for future testing of the role of VpreB selection on epitope recognition and antibody production. In Aim 1, we will create ?HC expression vectors with Y101-containing V domains cloned from living late preB cells that have passed preBCR selection, and V domains cloned from apoptotic early preB cells with disfavored amino acids at 101, such as leucine, that have failed preBCR selection. We will create mutated VpreB expression vectors with alanine (A), glycine (G) or valine (V) substitutions for RDR. These ?HC and mutated VpreB expression vectors, along with ?5, will be introduced into a preBCR negative proB cell line in which the formation and function of preBCR can be monitored by downstream signaling calcium flux. These in vitro studies will define residues in VpreB and CDR-H3 that are critical for optimal preBCR formation and function. In Aim 2, we will use CRISPR/Cas9 to selectively mutate VpreB RDR to A, G or V. We will examine patterns of cell cycling and apoptosis in developing B cells from mice with mutant VpreB. We will sequence VDJC? transcripts from living and apoptotic preB cells from these mice, and then assess sequence- structure correlations by molecular modeling. These in silico and in vivo studies will define the role of the VpreB CDR-H3 sensing site in preBCR formation, signaling, and checkpoint passage, and its effect on the preimmune repertoire and antibody functional activity. As per the developmental nature of this application, in future R01-funded studies these VpreB mutant mice will be used to test the role of preBCR selection in regulating epitope recognition and thus shaping the immune response to pathogens and self-antigens.